1. Field of the Invention
A novel ligand-mediated immunofunctional assay (LIFA) method is described for detecting the presence and quantitating the amount of a polypeptide hormone binding protein in a biological fluid and/or determining the amount of the ligand polypeptide hormone specifically bound to the hormone binding protein. This modified immunometric assay for a hormone binding protein uses: 1) a first solid phase bound antibody to capture the hormone binding protein; 2) a saturating amount of ligand hormone; and, 3) a labeled second antibody specific for the ligand hormone.
2. Description of the Background Art
A hormone binding protein (HBP) is a carrier protein found in biological fluids which has binding specificity for a ligand polypeptide hormone. Examples of such HBP's are growth hormone binding protein (GHBP), epidermal growth factor (EGF) binding protein, insulin-like growth factor 1 and 2 (IGF-1, IGF-2) binding proteins (five of them), platelet derived growth factor (PDGF) binding protein, nerve growth factor (NGF) binding protein, insulin binding protein, corticotropin releasing factor (CRF) binding protein, transforming growth factor beta (TGF-.beta.) binding protein and activin binding protein (Follistatin).
One of the best characterized polypeptide hormone binding proteins is the GHBP. The GHBP discussed in this invention is the extracellular domain of the GH receptor which circulates in blood and functions as a GHBP in several species (Ymer, S.I, and Herington, A.C., Mol. Cell. Endocrinol. 41:153 [1985]; Smith, W.C. and Talamantes, F., Endocrinology 123:1489-94 [1988]; Emtner, M., and Roos, P., Acta Endocrinologica [Copenhagen] 122:296-302 [1990]), including man (Baumann, G. et al., J. Clin. Endocrinol. Metab. 62:134-141 [1986]; Herington, A.C. et al., J. Clin. Invest. 77:1817-1823 [1986]). Little is known about the fate of the GHBP or its regulation in various physiological and pathological conditions.
Hormone binding proteins have been assayed by antibody based precipitation methods where the ligand is labeled and the antibody is specific for the binding protein itself. Monoclonal antibodies specific for human growth hormone binding protein (hGHBP) were used by Barnard et al., J. Endocrinol, 123 (2) p327-32 1989; for rabbit GHBP by Ymer et al., Endocrinology, 125 (2) p993-9, 1989; and mouse by Smith et al., Endocrinology 123, (3) p1489-94, 1988. Currently available methods for estimating GHBP levels in blood are based on incubation of the sample with radiolabeled GH, followed by separation of bound and free GH (Baumann, G. et al., Acta Endocrinologica (Copenhagen) 119:529-34 [1988]; Amit, T. et al., J. Clin. Endo. Metab. 71:474-479 [1990]). The results obtained by these assays are difficult to interpret due to interference by endogenous GH (Baumann, G. et al., J. Clin. Endocrinol. Metab. 62:134-141 [1986]). Others who have used labeled growth hormone to detect GHBP are: Emtner et al., Acta Endocrinol 122(3 ):296-302, (1990); Silbergeld et al., Clin. Endocrinol. 31(3):295-303 (1989); Daughaday et al., J. Clin. Endocrinol Metab., 65(5):1072-4, (1987); Herington et al., J. Clin. Invest. 77(6):1817-23, (1986); and Laron et al., Acta Endocrinol. 121(4):603-8 (1989). These assays for GHBP in blood have serious problems. They are laborious, requiring separation of complexed GHBP-GH by size-exclusion chromatography or antibody precipitation, and they may not give consistent results from one laboratory to another. In addition, they generate results that are arbitrary (i.e. not calibrated to a common protein standard) and influenced by endogenous growth hormone. Therefore, there is a need for an improved assay method which will allow detection of all the polypeptide hormone binding proteins, including those bound to endogenous polypeptide hormone.
A monoclonal antibody-based immunoradiometric assay for IGF binding protein was described by Pekonen et al, J. Immunoassay 10:325-37 (1989). Immunometric or sandwich immunoassays using high affinity monoclonal antibodies were taught in David et al., U.S. Pat. No. 4,486,530. Such sandwich assays have an antigen with two or more epitopes sandwiched between two antibodies. Circulating proteins that bind non-polypeptide hormone ligands, such as the thyroxine binding protein, have been measured using fluorescent labeled tracer (U.S. Pat. No. 4,476,228) in order to determine the number of binding protein sites not occupied by thyroxine. Iodothyronine immunoassays in a biological fluid using blocking agents and thyroxine binding globulin were described in Gordon et al, U.S. Pat. No. 4,622,293.
Specific binding pairs (SBP) are discussed in reference to antigen-antibody reactions, ligand-receptor, hormone-receptor and lectin-oligosaccharide (U.S. Pat. No. 4,956,302). Zuk et al., (U.S. Pat. No. 4,594,327) describes assays of whole blood to detect members of such SBPs wherein one member of the SBP must be attached to the solid phase prior to contacting the blood. The other second member of the SBP is detected using labeled second SBP member in competitive reactions. Similarly, Weng et al. (U.S. Pat. No. 4,737,456) describes a method of reducing interfering substances in assays of a SBP member wherein the individual member of the SBP is labeled. The receptor is used in a competitive assay to capture both labeled and unlabeled ligand, not to analyze for the presence of and quantify receptor as in the present invention.